Yuri Ivashchenko

Pharmacological Characterization of SAR407899, a Novel Rho-Kinase Inhibitor

Abstract—Recent advances in basic and clinical research have identified Rho kinase as an important target potentially implicated in a variety of cardiovascular diseases. Rho kinase is a downstream mediator of RhoA that leads to stress fiber formation, membrane ruffling, smooth muscle contraction, and cell motility. Increased Rho-kinase activity is associated with vasoconstriction and elevated blood pressure. We identified a novel inhibitor of Rho kinase (SAR407899) and characterized its effects in biochemical, cellular, tissue-based, and in vivo assays. SAR407899 is an ATP-competitive Rho-kinase inhibitor, equipotent against human and rat-derived Rho-kinase 2 with inhibition constant values of 36 nM and 41 nM, respectively. It is highly selective in panel of 117 receptor and enzyme targets. SAR407899 is ≈8-fold more active than fasudil. In vitro, SAR407899 demonstrated concentration-dependent inhibition of Rho-kinase-mediated phosphorylation of myosin phosphatase, thrombin-induced stress fiber formation, platelet-derived growth factor-induced proliferation, and monocyte chemotactic protein-1-stimulated chemotaxis. SAR407899 potently (mean IC50 values: 122 to 280 nM) and species-independently relaxed precontracted isolated arteries of different species and different vascular beds. In vivo, over the dose range 3 to 30 mg/kg PO, SAR407899 lowered blood pressure in a variety of rodent models of arterial hypertension. The antihypertensive effect of SAR407899 was superior to that of fasudil and Y-27632. In conclusion, SAR407899 is a novel and potent selective Rho-kinase inhibitor with promising antihypertensive activity.

Interleukin-1β stimulates acute phase response and C-reactive protein synthesis by inducing an NFκB- and C/EBPβ-dependent autocrine interleukin-6 loop

Cytokines interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) are involved in acute phase response (APR). C-reactive protein (CRP), the prototype acute phase protein, may represent an important component in the pathogenesis of arteriosclerosis and may also be a target for drug development. Inhibition of CRP synthesis is one potential strategy. Understanding CRP synthesis, however, is a prerequirement for the development of CRP-inhibitors. From studies in hepatoma cell lines, IL-1beta and IL-6 were considered as equal inductors of APR and CRP. We investigated IL-1beta- and IL-6-effects on primary human hepatocytes (PHH) and Hep3B-cells. Kupffer cell contamination in PHH preparations was <3%. In PHH, several APP like CRP, haptoglobin (HP), lipopolysaccharide-binding protein (LBP) or hepcidin (HAMP) were regulated similarly by IL-1beta and IL-6, though signal transduction pathways of these cytokines are different. In Hep3B-cells, APP were regulated exclusively by IL-6. IL-1beta induced IL-6-synthesis in PHH but not in Hep3B-cells. C/EBPbeta-overexpression in Hep3B-cells reconstituted IL-1beta-mediated IL-6/CRP inducibility. In PHH and in C/EBPbeta-overexpressing Hep3B-cells, neutralizing anti-IL-6-antibodies blocked IL-1beta-mediated APR. Inhibition of protein synthesis and NFkappaB-signalling blocked IL-1beta- but not IL-6-mediated CRP-expression in PHH, whereas Janus-Kinase-1-inhibition blocked IL-1beta- and IL-6-mediated APR. IL-1beta induces APR in PHH via an NFkappaB- and C/EBPbeta-dependent autocrine IL-6-loop. These findings partly reconcile the understanding of APR and may help to design a transcriptional suppressor of CRP for the treatment of cardiovascular disease.

Protein Kinase C Pathway Is Involved in Transcriptional Regulation of C-Reactive Protein Synthesis in Human Hepatocytes

Objective— C-Reactive protein (CRP) is the prototype acute phase protein and a cardiovascular risk factor. Interleukin-1&bgr; (IL-1&bgr;) and IL-6 stimulate CRP synthesis in hepatocytes. We searched for additional pathways regulating CRP expression. Methods and Results— Primary human hepatocytes (PHHs) were treated with IL-1&bgr;, IL-6, and protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBu). CRP was analyzed by quantitative RT-PCR and ELISA. PDBu significantly induced CRP transcription by 21.0±9.24-fold and protein release by 2.9±0.5-fold. Transcriptional regulation was studied in detail in hepatoma G2 (HepG2) cells stably transfected with the 1-kb CRP promoter (HepG2–ABEK14 cells). In these cells, PDBu significantly induced CRP transcription by 5.39±0.66-fold. Competetive inhibition with bisindolylmaleimide derivative LY333531 abolished PDBu-mediated promoter activation. Competetive inhibition with I&kgr;B kinase inhibitor I229 also inhibited PDBu effects. Importantly, IL-8 significantly induced CRP release in PHHs by 58.675±19.1-fold, which was blockable by LY333531. Conclusions— This study describes a novel PKC-dependent transcriptional regulation of CRP gene expression, which, in analogy to the classical IL-1&bgr; and IL-6 pathways, is operational in hepatocytes only. It also identifies IL-8 as a potential physiological PKC activator. HepG2–ABEK14 cells may be useful for high throughput screening to identify inhibitors of CRP synthesis for the prevention of cardiovascular disease.

Synthesis and Characterization of a Promising Novel FFAR1/GPR40 Targeting Fluorescent Probe for β-Cell Imaging

Diabetes affects an increasing number of patients worldwide and is responsible for a significant rise in healthcare expenses. Imaging of β-cells bears the potential to contribute to an improved understanding, diagnosis, and development of new treatment options for diabetes. Here, we describe the first small molecule fluorescent probe targeting the free fatty acid receptor 1 (FFAR1/GPR40). This receptor is highly expressed on β-cells, and was up to now unexplored for imaging purposes. We designed a novel probe by facile modification of the selective and potent FFAR1 agonist TAK-875. Effective and specific binding of the probe was demonstrated using FFAR1 overexpressing cells. We also successfully labeled FFAR1 on MIN6 and INS1E cells, two widely used β-cell models, by applying an effective amplification protocol. Finally, we showed that the probe is capable of inducing insulin secretion in a glucose-dependent manner, thus demonstrating that functional activity of the probe was maintained. These results suggest that our probe represents a first important step to successful β-cell imaging by targeting FFAR1. The developed probe may prove to be particularly useful for in vitro and ex vivo studies of diabetic cellular and animal models to gain new insights into disease pathogenesis.

End-organ protection in hypertension by the novel and selective Rho-kinase inhibitor, SAR407899

AIM To compare the therapeutic efficacy of SAR407899 with the current standard treatment for hypertension [an angiotensin converting enzyme (ACE)-inhibitor and a calcium channel blocker] and compare the frequency and severity of the hypertension-related end-organ damage. METHODS Long-term pharmacological characte-rization of SAR407899 has been performed in two animal models of hypertension, of which one is sensitive to ACE-inhibition (LNAME) and the other is insensitive [deoxycorticosterone acetate (DOCA)]. SAR407899 efficiently lowered high blood pressure and significantly reduced late-stage end organ damage as indicated by improved heart, kidney and endothelial function and reduced heart and kidney fibrosis in both models of chronic hypertension. RESULTS Long term treatment with SAR407899 has been well tolerated and dose-dependently reduced elevated blood pressure in both models with no signs of tachyphylaxia. Blood pressure lowering effects and protective effects on hypertension related end organ damage of SAR407899 were superior to ramipril and amlodipine in the DOCA rat. Typical end-organ damage was significantly reduced in the SAR407899-treated animals. Chronic administration of SAR407899 significantly reduced albuminuria in both models. The beneficial effect of SAR407899 was associated with a reduction in leukocyte/macrophage tissue infiltration. The overall protective effect of SAR407899 was superior or comparable to that of ACE-inhibition or calcium channel blockade. Chronic application of SAR407899 protects against hypertension and hypertension-induced end organ damage, regardless of the pathophysiological mechanism of hypertension. CONCLUSION Rho-kinases-inhibition by the SAR407899 represents a new therapeutic option for the treatment of hypertension and its complications.